120 PTEROYLGLUTAMIC ACID 



with boiling pH 4.0 buffer, anhydroleucovorin-B (XIX)''"' ^^ is formed; the 

 exact structure of this derivative has not been completely elucidated. Both 

 anhydroleucovorin-A and anhydroleucovorin-B can be converted to iso- 

 leucovorin chloride with 0.1 A^ hydrochloric acid and to leucovorin with 

 alkali under anaerobic conditions. Another derivative is 5,10-diformyl- 

 5,6,7,8-tetrahydro PGA (XVII),"' ^* which can be formed from either 

 5,6,7,8-tetrahydro PGA (XVI), 10-formyl-5,6,7,8-tetrahydro PGA 

 (XIV), or leucovorin (XII) by formylation with 98 to 100% formic acid in 

 the presence of acetic anhydride; this substance has an activity for Le. 

 citrovorum which is equivalent to 0.5 % of pure leucovorin. Alkaline hy- 

 drolysis removes the 10-formyl group to give leucovorin. 



It is conceivable that, during migration of the formyl group which occurs 

 in the synthesis of leucovorin and its transformation products, an inter- 

 mediate hydroxymethylene bridge might result. Two stereoisomeric con- 

 figurations are possible for such a bridge. Although many have claimed^'^'^^ 

 that the formyl and other acyl derivatives of aromatic /3-secondary diamines 

 exist as ring structures with hydroxymethylene bridges, recent results ob- 

 tained by infra-red absorption studies of some acyl compounds of similar 

 structure indicate that the open amide form exists at least to some extent.'"" 



e. Relation of Natural Citrovorum Factor to Leucovorin 



Recently Keresztesy and Silverman^"^ and Sauberlich'"^ have succeeded 

 in isolating crystalline citrovorum factor from liver which has permitted 

 comparison of the properties of the naturally occurring vitamin with 

 5-formyl-5,6,7,8-tetrahydro PGA. The product obtained from horse liver 

 by Keresztesy and Silverman was found to be approximately twice as active 

 as synthetic 5-f ormyl-5 , 6 , 7 , 8-tetrahydro PGA for growth of Leuconostoc 

 citrovorum. In 0.1 A^ NaOH solution (at a concentration of 10 mg. per liter) 

 leucovorin exhibits a maximum at 282 ran (optical density — 0.631) and a 

 minimum at 243 ran (optical density = 0.123). When the two compounds 

 are examined spectrophotometrically under identical conditions, they are 

 indistinguishable. Since the natural factor is twice as active microbio- 

 logically as the synthetic product, and since there appears to be no spectral 

 difference between the compounds, it is possible that the synthetic factor 

 is a mixture of diastereoisomers, one of which is the biologically active, 

 naturally occurring form. Examination of the structure of 5-f ormyl-5, 6,7,8- 

 tetrahydro PGA indicates the possibility of diastereoisomers. In the synthe- 



" O. Fischer, Ber. 34, 930 (1901). 



«8 St. Niementowski, Ber. 20, 1874 (1887). 



»« O. Fischer and M. Rigaud, Ber. 34, 4203 (1901); 35, 1258 (1902). 

 '00 C. W. Smith, R. S. Rasmussen, and S. A. Balhird, J. Am. Chem. Soc. 71, 1082 (1949) . 

 '01 J. C. Keresztesy and M. Silverman, /. Am. Chem. Soc. 73, 5510 (1951). 

 '02 H. E. Sauberlich, J. Biol. Chem. 195, 337 (1952). 



